Bt toxin receptors from lepidopteran insects

ABSTRACT

The invention relates to Bt toxin resistance management. The invention particularly relates to the isolation and characterization of nucleic acid and polypeptides for a novel Bt toxin receptor. The nucleic acid and polypeptides are useful in identifying and designing novel Bt toxin receptor ligands including novel insecticidal toxins.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. application Ser.No. 09/715,909, filed Nov. 17, 2000, now U.S. Pat. No. 7,060,491, whichclaims the benefit of U.S. Provisional Application Ser. No. 60/234,099,filed Sep. 21, 2000 and U.S. Provisional Application Ser. No.60/166,285, filed Nov. 18, 1999, the contents of which are hereinincorporated by reference in their entirety.

FIELD OF THE INVENTION

The field of the invention is manipulating Bt toxin susceptibility inplant pests. The field of the invention relates to the isolation andcharacterization of nucleic acid and polypeptides for a novel Bt toxinreceptor. The nucleic acid and polypeptides are useful in developing newinsecticides.

BACKGROUND OF THE INVENTION

Traditionally, growers used chemical pesticides as a means to controlagronomically important pests. The introduction of transgenic plantscarrying the delta-endotoxin from Bacillus thuringiensis (Bt) afforded anon-chemical method of control. Bt toxins have traditionally beencategorized by their specific toxicity towards specific insectcategories. For example, the Cryl group of toxins are toxic toLepidoptera. The Cryl group includes, but is not limited to, CrylA(a),CrylA(b) and CrylA(c). See Hofte et al (1989) Microbiol Rev 53: 242-255.

Lepidopteran insects cause considerable damage to maize crops throughoutNorth America and the world. One of the leading pests is Ostrinianubilalis, commonly called the European Corn Borer (ECB). Genes encodingthe crystal proteins CrylA(b) and CrylA(c) from Bt have been introducedinto maize as a means of ECB control. These transgenic maize hybridshave been effective in control of ECB. However, developed resistance toBt toxins presents a challenge in pest control. See McGaughey et al.(1998) Nature Biotechnology 16: 144-146; Estruch et al. (1997) NatureBiotechnology 15:137-141; Roush et al. (1997) Nature Biotechnology 15816-817; and Hofte et al (1989) Microbiol Rev 53: 242-255.

The primary site of action of Cryl toxins is in the brush bordermembranes of the midgut epithelia of susceptible insect larvae such aslepidopteran insects. CrylA toxin binding polypeptides have beencharacterized from a variety of Lepidopteran species. A CrylA(c) bindingpolypeptide with homology to an aminopeptidase N has been reported fromManduca sexta, Lymantria dispar, Helicoverpa zea and Heliothisvirescens. See Knight et al (1994) Mol Micro 11: 429-436; Lee et al.(1996) Appl Environ Micro 63: 2845-2849; Gill et al. (1995) J. Biol.Chem 270: 27277-27282; and Garczynski et al. (1991) Appl EnvironMicrobiol 10: 2816-2820.

Another Bt toxin binding polypeptide (BTR1) cloned from M. sexta hashomology to the cadherin polypeptide superfamily and binds CrylA(a),CrylA(b) and CrylA(c). See Vadlamudi et al. (1995) J Biol Chem270(10):5490-4, Keeton et al. (1998) Appl Environ Microbiol64(6):2158-2165; Keeton et al. (1997) Appl Environ Microbiol63(9):3419-3425 and U.S. Pat. No. 5,693,491.

A subsequently cloned homologue to BTR1 demonstrated binding to CrylA(a)from Bombyx mori as described in Ihara et al. (1998) ComparativeBiochemistry and Physiology, Part B 120:197-204 and Nagamatsu et al.(1998) Biosci. Biotechnol. Biochem. 62(4):727-734.

Identification of the plant pest binding polypeptides for Bt toxins areuseful for investigating Bt toxin-Bt toxin receptor interactions,selecting and designing improved toxins, developing novel insecticides,and new Bt toxin resistance management strategies.

SUMMARY OF THE INVENTION

Compositions and methods for modulating susceptibility of a cell to Bttoxins are provided. The compositions include Bt toxin receptorpolypeptides, and fragments and variants thereof, from the lepidopteraninsects European corn borer (ECB, Ostrinia nubilalis), corn earworm(CEW, Heliothis Zea), and fall armyworm (FAW, Spodoptera frugiperda).The polypeptides bind CrylA toxins, more particularly CrylA(b). Nucleicacids encoding the polypeptides, antibodies specific to thepolypeptides, as well as nucleic acid constructs for expressing thepolypeptides in cells of interest are also provided.

The methods are useful for investigating the structure-functionrelationships of Bt toxin receptors; investigating the toxin-receptorinteractions; elucidating the mode of action of Bt toxins; screening andidentifying novel Bt toxin receptor ligands including novel insecticidaltoxins; and designing and developing novel Bt toxin receptor ligands.

The methods are useful for managing Bt toxin resistance in plant pests,and protecting plants against damage by plant pests.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically depicts the location of the signal sequence,putative glycosilation sites, cadherin-like domains, transmembranesegment, CrylA binding region and protein kinase C phosphorylation siteof the Bt toxin receptor from Ostrinia nubilalis; the nucleotidesequence of the receptor set forth in SEQ ID NO: 1 and the correspondingdeduced amino acid sequence in SEQ ID NO:2.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to novel receptor polypeptides that bind Bttoxin, the receptor being derived from the order lepidoptera. Thereceptors of the invention include those receptor polypeptides that bindBt toxin and are derived from the lepidopteran superfamily Pyraloideaand particularly from the species Ostrinia, specifically Ostrinianubilalis; those derived from Spodoptera frugiperda (S. frugperda); andthose derived from Heliothus Zea (H. Zea). The polypeptides havehomology to members of the cadherin superfamily of proteins.Accordingly, compositions of the invention include isolated polypeptidesthat are involved in Bt toxin binding. In particular, the presentinvention provides for isolated nucleic acid molecules comprisingnucleotide sequences encoding the amino acid sequences shown in SEQ IDNOs: 2, 4, and 6; or the nucleotide sequences having the DNA sequencesdeposited in a plasmid in a bacterial host as Patent Deposit No.PTA-278, PTA-1760, and PTA-2222. Further provided are polypeptideshaving an amino acid sequence encoded by a nucleic acid moleculedescribed herein, for example those set forth in SEQ ID NOs: 1, 3, and5; those deposited in a plasmid in a bacterial host as Patent DepositNos. PTA-278, PTA-1760, and PTA-2222; and fragments and variantsthereof.

Plasmids containing the nucleotide sequences of the invention weredeposited with the Patent Depository of the American Type CultureCollection (ATCC), Manassas, Va. on Jun. 25, 1999; Apr. 25, 2000; andJul. 11, 2000; and assigned Patent Deposit Nos. PTA-278, PTA-1760, andPTA-2222. These deposits will be maintained under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure. These deposits weremade merely as a convenience for those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. §112.

The term “nucleic acid” refers to all forms of DNA such as cDNA orgenomic DNA and RNA such as mRNA, as well as analogs of the DNA or RNAgenerated using nucleotide analogs. The nucleic acid molecules can besingle stranded or double stranded. Strands can include the coding ornon-coding strand.

The invention encompasses isolated or substantially purified nucleicacid or polypeptide compositions. An “isolated” or “purified” nucleicacid molecule or polypeptide, or biologically active portion thereof, issubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. Preferably,an “isolated” nucleic acid is free of sequences (preferably polypeptideencoding sequences) that naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated nucleic acid molecule cancontain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kbof nucleotide sequences that naturally flank the nucleic acid moleculein genomic DNA of the cell from which the nucleic acid is derived. Apolypeptide that is substantially free of cellular material includespreparations of polypeptide having less than about 30%, 20%, 10%, 5%,(by dry weight) of contaminating polypeptide. When the polypeptide ofthe invention or biologically active portion thereof is recombinantlyproduced, preferably culture medium represents less than about 30%, 20%,10%, or 5% (by dry weight) of chemical precursors ornon-polypeptide-of-interest chemicals.

It is understood, however, that there are embodiments in whichpreparations that do not contain the substantially pure polypeptide mayalso be useful. Thus, less pure preparations can be useful where thecontaminating material does not interfere with the specific desired useof the peptide. The compositions of the invention also encompassfragments and variants of the disclosed nucleotide sequences and thepolypeptides encoded thereby.

The compositions of the invention are useful for, among other uses,expressing the receptor polypeptides in cells of interest to producecellular or isolated preparations of the polpeptides for investigatingthe structure-function relationships of Bt toxin receptors;investigating the toxin-receptor interactions; elucidating the mode ofaction of Bt toxins; screening and identifying novel Bt toxin receptorligands including novel insecticidal toxins; and designing anddeveloping novel Bt toxin receptor ligands including novel insecticidaltoxins.

The isolated nucleotide sequences encoding the receptor polypeptides ofthe invention are expressed in a cell of interest; and the Bt toxinreceptor polypeptides produced by the expression is utilized in intactcell or in-vitro receptor binding assays, and/or intact cell toxicityassays. Methods and conditions for Bt toxin binding and toxicity assaysare known in the art and include but are not limited to those describedin U.S. Pat. No. 5,693,491; T. P. Keeton et al. (1998) Appl. Environ.Microbiol. 64(6):2158-2165; B. R. Francis et al. (1997) Insect Biochem.Mol. Biol. 27(6):541-550; T. P. Keeton et al. (1997) Appl. Environ.Microbiol. 63(9):3419-3425; R. K. Vadlamudi et al. (1995) J. Biol. Chem.270(10):5490-5494; Ihara et al. (1998) Comparative Biochem. Physiol. B120:197-204; Nagamatsu et al. (1998) Biosci. Biotechnol. Biochem.62(4):727-734, herein incorporated by reference. Such methods could bemodified by one of ordinary skill in the art to develop assays utilizingthe polypeptides of the invention.

By “cell of interest” is intended any cell in which expression of thepolypeptides of the invention is desired. Cells of interest include, butare not limited to mammalian, avian, insect, plant, bacteria, fungi andyeast cells. Cells of interest include but are not limited to culturedcell lines, primary cell cultures, cells in vivo, and cells oftransgenic organisms.

The methods of the invention encompass using the polypeptides encoded bythe nucleotide sequences of the invention in receptor binding and/ortoxicity assays to screen candidate ligands and identify novel Bt toxinreceptor ligands, including receptor agonists and antagonists. Candidateligands include molecules available from diverse libraries of smallmolecules created by combinatorial synthetic methods. Candidate ligandsalso include, but are not limited to antibodies, peptides, and othersmall molecules designed or deduced to interact with the receptorpolypeptides of the invention. Candidate ligands include but are notlimited to peptide fragments of the receptor, anti-receptor antibodies,antiidiotypic antibodies mimicking one or more receptor binding domainsof a toxin, fusion proteins produced by combining two or more toxins orfragments thereof, and the like. Ligands identified by the screeningmethods of the invention include potential novel insecticidal toxins,the insecticidal activity of which can be determined by known methods;for example, as described in U.S. Pat. No. 5,407,454; U.S. applicationSer. No. 09/218,942; U.S. application Ser. No. 09/003,217.

The invention provides methods for screening for ligands that bind tothe polypeptides described herein. Both the polypeptides and relevantfragments thereof (for example, the toxin binding domain) can be used toscreen by assay for compounds that bind to the receptor and exhibitdesired binding characteristics. Desired binding characteristicsinclude, but are not limited to binding affinity, binding sitespecificity, association and dissociation rates, and the like. Thescreening assays could be intact cell or in vitro assays which includeexposing a ligand binding domain to a sample ligand and detecting theformation of a ligand-binding polypeptide complex. The assays could bedirect ligand-receptor binding assays or ligand competition assays.

In one embodiment, the methods comprise providing at least one Bt toxinreceptor polypeptide of the invention, contacting the polypeptide with asample and a control ligand under conditions promoting binding; anddetermining binding characteristics of sample ligands, relative tocontrol ligands. The methods encompass any method known to the skilledartisan which can be used to provide the polypeptides of the inventionin a binding assay. For in vitro binding assays, the polypeptide may beprovided as isolated, lysed, or homogenized cellular preparations.Isolated polypeptides may be provided in solution, or immobilized to amatrix. Methods for immobilizing polypeptides are well known in the art,and include but are not limited to construction and use of fusionpolypeptides with commercially available high affinity ligands. Forexample, GST fusion proteins can be adsorbed onto glutathione sepharosebeads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatizedmicrotitre plates. The polypeptides can also be immobilized utilizingwell techniques in the art utilizing conjugation of biotin andstreptavidin. The polypeptides can also be immobilized utilizing wellknown techniques in the art utilizing chemical conjugation (linking) ofpolypeptides to a matrix. Alternatively, the polypeptides may beprovided in intact cell binding assays in which the polypeptides aregenerally expressed as cell surface Bt toxin receptors.

The invention provides methods utilizing intact cell toxicity assays toscreen for ligands that bind to the receptor polypeptides describedherein and confer toxicity upon a cell of interest expressing thepolypeptide. A ligand selected by this screening is a potentialinsecticidal toxin to insects expressing the receptor polypeptides,particularly enterally. This deduction is premised on theories thatinsect specificity of a particular Bt toxin is determined by thepresence of the receptor in specific insect species, or that binding ofthe toxins is specific for the receptor of some insect species and isbind is insignificant or nonspecific for other variant receptors. See,for example Hofte et al (1989) Microbiol Rev 53: 242-255. The toxicityassays include exposing, in intact cells expressing a polypeptide of theinvention, the toxin binding domain of the polypeptide to a sampleligand and detecting the toxicity effected in the cell expressing thepolypeptide. By “toxicity” is intended the decreased viability of acell. By “viability” is intended the ability of a cell to proliferateand/or differentiate and/or maintain its biological characteristics in amanner characteristic of that cell in the absence of a particularcytotoxic agent.

In one embodiment, the methods of the present invention compriseproviding at least one cell surface Bt toxin receptor polypeptide of theinvention comprising an extracellular toxin binding domain, contactingthe polypeptide with a sample and a control ligand under conditionspromoting binding, and determining the viability of the cell expressingthe cell surface Bt toxin receptor polypeptide, relative to the controlligand.

By “contacting” is intended that the sample and control agents arepresented to the intended ligand binding site of the polypeptides of theinvention.

By “conditions promoting binding” is intended any combination ofphysical and biochemical conditions that enables a ligand of thepolypeptides of the invention to determinably bind the intendedpolypeptide over background levels. Examples of such conditions forbinding of Cryl toxins to Bt toxin receptors, as well as methods forassessing the binding, are known in the art and include but are notlimited to those described in Keeton et al. (1998) Appl EnvironMicrobiol 64(6): 2158-2165; Francis et al. (1997) Insect Biochem MolBiol 27(6):541-550; Keeton et al. (1997) Appl Environ Microbiol63(9):3419-3425; Vadlamudi et al. (1995) J Biol Chem 270(10):5490-5494;Ihara et al. (1998) Comparative Biochemistry and Physiology, Part B120:197-204; and Nagamatsu et al. (1998) Biosci. Biotechnol. Biochem.62(4):727-734, the contents of which are herein incorporated byreference. In this aspect of the present invention, known andcommercially available methods for studying protein-proteininteractions, such as yeast and/or bacterial two-hybrid systems couldalso be used. Two-hybrid systems are available from, for example,CLONTECH (Palo Alto, Calif.) or Display Systems Biotech Inc. (Vista,Ca).

The compositions and screening methods of the invention are useful fordesigning and developing novel Bt toxin receptor ligands including novelinsecticidal toxins. Various candidate ligands; ligands screened andcharacterized for binding, toxicity, and species specificity; and/orligands having known characteristics and specificities, could be linkedor modified to produce novel ligands having particularly desiredcharacteristics and specificities. The methods described herein forassessing binding, toxicity and insecticidal activity could be used toscreen and characterize the novel ligands.

In one embodiment of the present invention, the sequences encoding thereceptors of the invention, and variants and fragments thereof, are usedwith yeast and bacterial two-hybrid systems to screen for Bt toxins ofinterest (for example, more specific and/or more potent toxins), or forinsect molecules that bind the receptor and can be used in developingnovel insecticides.

By “linked” is intended that a covalent bond is produced between two ormore molecules. Known methods that can be used for modification and/orlinking of polypeptide ligands such as toxins, include but are notlimited to mutagenic and recombinogenic approaches including but notlimited to site-directed mutagenesis, chimeric polypeptide constructionand DNA shuffling. Such methods are described in further detail below.Known polypeptide modification methods also include methods for covalentmodification of polypeptides. “Operably linked” means that the linkedmolecules carry out the function intended by the linkage.

The compositions and screening methods of the present invention areuseful for targeting ligands to cells expressing the receptorpolypeptides of the invention. For targeting, secondary polyeptides,and/or small molecules which do not bind the receptor polypeptides ofthe invention are linked with one or more primary ligands which bind thereceptor polypeptides; including but not limited to Cryl A toxin; moreparticularly Cryl A(b) toxin or a fragment thereof. By this linkage, anypolypeptide and/or small molecule linked to a primary ligand could betargeted to the receptor polypeptide, and thereby to a cell expressingthe receptor polypeptide; wherein the ligand binding site is availableat the extracellular surface of the cell.

In one embodiment of the invention, at least one secondary polypeptidetoxin is linked with a primary Cryl A toxin capable of binding thereceptor polypeptides of the invention to produce a combination toxinwhich is targeted and toxic to insects expressing the receptor for theprimary toxin. Such insects include those of the order lepidoptera,superfamily Pyraloidea and particularly from the species Ostrinia,specifically Ostrinia nubilalis. Such insects include the lepidopteransS. frugiperda and H. Zea. Such a combination toxin is particularlyuseful for eradicating or reducing crop damage by insects which havedeveloped resistance to the primary toxin.

For expression of the Bt toxin receptor polypeptides of the invention ina cell of interest, the Bt toxin receptor sequences are provided inexpression cassettes. The cassette will include 5′ and 3′ regulatorysequences operably linked to a Bt toxin receptor sequence of theinvention. In this aspect of the present invention, by “operably linked”is intended a functional linkage between a promoter and a secondsequence, wherein the promoter sequence initiates and mediatestranscription of the DNA sequence corresponding to the second sequence.In reference to nucleic acids, generally, operably linked means that thenucleic acid sequences being linked are contiguous and, where necessaryto join two polypeptide coding regions, contiguous and in the samereading frame. The cassette may additionally contain at least oneadditional gene to be cotransformed into the organism. Alternatively,the additional gene(s) can be provided on multiple expression cassettes.

Such an expression cassette is provided with a plurality of restrictionsites for insertion of the Bt toxin receptor sequence to be under thetranscriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, aBt toxin receptor nucleotide sequence of the invention, and atranscriptional and translational termination region functional in hostcells. The transcriptional initiation region, the promoter, may benative or analogous, or foreign or heterologous to the plant host.Additionally, the promoter may be the natural sequence or alternativelya synthetic sequence. By “foreign” is intended that the transcriptionalinitiation region is not found in the native host cells into which thetranscriptional initiation region is introduced. As used herein, achimeric gene comprises a coding sequence operably linked to atranscription initiation region that is heterologous to the codingsequence.

While it may be preferable to express the sequences using heterologouspromoters, the native promoter sequences may be used. Such constructswould change expression levels of Bt toxin receptor in the cell ofinterest. Thus, the phenotype of the cell is altered.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,or may be derived from another source.

Where appropriate, the gene(s) may be optimized for increased expressionin a particular transformed cell of interest. That is, the genes can besynthesized using host cell-preferred codons for improved expression.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5′ leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translation leaders are known in the art andinclude: picomavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize DwarfMosaic Virus); Virology 154:9-20), and human immunoglobulin heavy-chainbinding polypeptide (BiP), (Macejak et al. (1991) Nature 353:90-94);untranslated leader from the coat polypeptide mRNA of alfalfa mosaicvirus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobaccomosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology ofmRNA, ed. Cech (Liss, New York), pp. 237-256); and maize chloroticmottle virus leader (MCMV) (Lommel et al. (1991) Virology 81:382-385).See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968. Othermethods known to enhance translation can also be utilized, for example,introns, and the like.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

Using the nucleic acids of the present invention, the polypeptides ofthe invention could be expressed in any cell of interest, the particularchoice of the cell depending on factors such as the level of expressionand/or receptor activity desired. Cells of interest include, but are notlimited to conveniently available mammalian, plant, insect, bacteria,and yeast host cells. The choice of promoter, terminator, and otherexpression vector components will also depend on the cell chosen. Thecells produce the protein in a non-natural condition (e.g., in quantity,composition, location, and/or time), because they have been geneticallyaltered through human intervention to do so.

It is expected that those of skill in the art are knowledgeable in thenumerous expression systems available for expression of a nucleic acidencoding a protein of the present invention. No attempt to describe indetail the various methods known for the expression of proteins inprokaryotes or eukaryotes will be made.

In brief summary, the expression of isolated nucleic acids encoding aprotein of the present invention will typically be achieved by operablylinking, for example, the DNA or cDNA to a promoter, followed byincorporation into an expression vector. The vectors can be suitable forreplication and integration in either prokaryotes or eukaryotes. Typicalexpression vectors contain transcription and translation terminators,initiation sequences, and promoters useful for regulation of theexpression of the DNA encoding a protein of the present invention. Toobtain high level expression of a cloned gene, it is desirable toconstruct expression vectors which contain, at the minimum, a strongpromoter to direct transcription, a ribosome binding site fortranslational initiation, and a transcription/translation terminator.One of skill would recognize that modifications can be made to a proteinof the present invention without diminishing its biological activity.Some modifications may be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, a methionine added at the amino terminus to provide aninitiation site, or additional amino acids (e.g., poly His) placed oneither terminus to create conveniently located restriction sites ortermination codons or purification sequences.

Prokaryotic cells may be used as hosts for expression. Prokaryotes mostfrequently are represented by various strains of E. coli; however, othermicrobial strains may also be used. Commonly used prokaryotic controlsequences which are defined herein to include promoters fortranscription initiation, optionally with an operator, along withribosome binding site sequences, include such commonly used promoters asthe beta lactamase (penicillinase) and lactose (lac) promoter systems(Chang et al. (1977) Nature 198:1056), the tryptophan (trp) promotersystem (Goeddel et al. (1980) Nucleic Acids Res. 8:4057) and thelambda-derived P L promoter and N-gene ribosome binding site (Shimatakeet al. (1981) Nature 292:128). The inclusion of selection markers in DNAvectors transfected in E. coli is also useful. Examples of such markersinclude genes specifying resistance to ampicillin, tetracycline, orchloramphenicol.

The vector is selected to allow introduction into the appropriate hostcell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for expressing a protein of the present invention areavailable using Bacillus sp. and Salmonella (Palva et al. (1983) Gene22:229-235; Mosbach et al. (1983) Nature 302:543-545).

A variety of eukaryotic expression systems such as yeast, insect celllines, plant and mammalian cells, are known to those of skill in theart. The sequences of the present invention can be expressed in theseeukaryotic systems. In some embodiments, transformed/transfected plantcells are employed as expression systems for production of the proteinsof the instant invention.

Synthesis of heterologous proteins in yeast is well known. Sherman, F.et al. (1982) Methods in Yeast Genetics, Cold Spring Harbor Laboratoryis a well recognized work describing the various methods available toproduce the protein in yeast. Two widely utilized yeast for productionof eukaryotic proteins are Saccharomyces cerevisia and Pichia pastoris.Vectors, strains, and protocols for expression in Saccharomyces andPichia are known in the art and available from commercial suppliers(e.g., Invitrogen). Suitable vectors usually have expression controlsequences, such as promoters, including 3-phosphoglycerate kinase oralcohol oxidase, and an origin of replication, termination sequences andthe like as desired.

A protein of the present invention, once expressed, can be isolated fromyeast by lysing the cells and applying standard protein isolationtechniques to the lysates. The monitoring of the purification processcan be accomplished by using Western blot techniques or radioimmunoassayor other standard immunoassay techniques.

The sequences encoding proteins of the present invention can also beligated to various expression vectors for use in transfecting cellcultures of, for instance, mammalian, insect, or plant origin.Illustrative of cell cultures useful for the production of the peptidesare mammalian cells. Mammalian cell systems often will be in the form ofmonolayers of cells although mammalian cell suspensions may also beused. A number of suitable host cell lines capable of expressing intactproteins have been developed in the art, and include the COS, HEK293,BHK21, and CHO cell lines. Expression vectors for these cells caninclude expression control sequences, such as an origin of replication,a promoter (e.g., the CMV promoter, a HSV tk promoter orpgk(phosphoglycerate kinase promoter)), an enhancer (Queen et al. (1986)Immunol. Rev. 89:49), and necessary processing information sites, suchas ribosome binding sites, RNA splice sites, polyadenylation sites(e.g., an SV40 large T Ag poly A addition site), and transcriptionalterminator sequences. Other animal cells useful for production ofproteins of the present invention are available, for instance, from theAmerican Type Culture Collection Catalogue of Cell Lines and Hybridomas(7th edition, 1992). A particular example of mammalian cells forexpression of a Bt toxin receptor and assessing Bt toxin cytotoxicitymediated by the receptor, includes embryonic 293 cells. See U.S. Pat.No. 5,693,491, herein incorporated by reference.

Appropriate vectors for expressing proteins of the present invention ininsect cells are usually derived from the SF9 baculovirus. Suitableinsect cell lines include mosquito larvae, silkworm, armyworm, moth andDrosophila cell lines such as a Schneider cell line (See Schneider etal. (1987) J Embryol. Exp. Morphol. 27: 353-365).

As with yeast, when higher animal or plant host cells are employed,polyadenylation or transcription terminator sequences are typicallyincorporated into the vector. An example of a terminator sequence is thepolyadenylation sequence from the bovine growth hormone gene. Sequencesfor accurate splicing of the transcript may also be included. An exampleof a splicing sequence is the VP1 intron from SV40 (Sprague et al.(1983) J. Virol. 45:773-781). Additionally, gene sequences to controlreplication in the host cell may be incorporated into the vector such asthose found in bovine papilloma virus-type vectors. Saveria-Campo, M.,Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector in DNA CloningVol. II a Practical Approach, D. M. Glover, ed., IRL Pres, Arlington,Va. pp. 213-238 (1985).

In a particular embodiment of the invention, it may be desirable tonegatively control receptor binding; particularly, when toxicity to acell is no longer desired or if it is desired to reduce toxicity to alower level. In this case, ligand-receptor polypeptide binding assayscan be used to screen for compounds which bind to the receptor but donot confer toxicity to a cell expressing the receptor. The examples of amolecule that can be used to block ligand binding include an antibodythat specifically recognizes the ligand binding domain of the receptorsuch that ligand binding is decreased or prevented as desired.

In another embodiment, receptor polypeptide expression could be blockedby the use of antisense molecules directed against receptor RNA orribozymes specifically targeted to this receptor RNA. It is recognizedthat with the provided nucleotide sequences, antisense constructions,complementary to at least a portion of the messenger RNA (mRNA) for theBt toxin receptor sequences can be constructed. Antisense nucleotidesare constructed to hybridize with the corresponding mRNA. Modificationsof the antisense sequences may be made as long as the sequenceshybridize to and interfere with expression of the corresponding mRNA. Inthis manner, antisense constructions having 70%, preferably 80%, morepreferably 85% sequence similarity to the corresponding antisensedsequences may be used. Furthermore, portions of the antisensenucleotides may be used to disrupt the expression of the target gene.Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200nucleotides, or greater may be used.

Fragments and variants of the disclosed nucleotide sequences andpolypeptides encoded thereby are encompassed by the present invention.By “fragment” is intended a portion of the nucleotide sequence, or aportion of the amino acid sequence, and hence a portion of thepolypeptide encoded thereby. Fragments of a nucleotide sequence mayencode polypeptide fragments that retain the biological activity of thenative polypeptide and, for example, bind Bt toxins. Alternatively,fragments of a nucleotide sequence that are useful as hybridizationprobes generally do not encode fragment polypeptides retainingbiological activity. Thus, fragments of a nucleotide sequence may rangefrom at least about 20 nucleotides, about 50 nucleotides, about 100nucleotides, and up to the full-length nucleotide sequence encoding thepolypeptides of the invention.

A fragment of a Bt toxin receptor nucleotide sequence that encodes abiologically active portion of a Bt toxin receptor polypeptide of theinvention will encode at least 15, 25, 30, 50, 100, 150, 200 or 250contiguous amino acids, or up to the total number of amino acids presentin a full-length Bt toxin receptor polypeptide of the invention (forexample, 1717, 1730, and 1734 amino acids for SEQ ID NOs:2, 4, and 6,respectively. Fragments of a Bt toxin receptor nucleotide sequence thatare useful as hybridization probes for PCR primers generally need notencode a biologically active portion of a Bt toxin receptor polypeptide.

Thus, a fragment of a Bt toxin receptor nucleotide sequence may encode abiologically active portion of a Bt toxin receptor polypeptide, or itmay be a fragment that can be used as a hybridization probe or PCRprimer using methods disclosed below. A biologically active portion of aBt toxin receptor polypeptide can be prepared by isolating a portion ofone of the Bt toxin receptor nucleotide sequences of the invention,expressing the encoded portion of the Bt toxin receptor polypeptide(e.g., by recombinant expression in vitro), and assessing the activityof the encoded portion of the Bt toxin receptor polypeptide. Nucleicacid molecules that are fragments of a Bt toxin receptor nucleotidesequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300,or 1,400 nucleotides, or up to the number of nucleotides present in afull-length Bt toxin receptor nucleotide sequence disclosed herein (forexample, 5498, 5527, and 5614 nucleotides for SEQ ID NOs: 1, 3, and 5,respectively).

By “variants” is intended substantially similar sequences. Fornucleotide sequences, conservative variants include those sequencesthat, because of the degeneracy of the genetic code, encode the aminoacid sequence of one of the Bt toxin receptor polypeptides of theinvention. Naturally occurring allelic variants such as these can beidentified with the use of well-known molecular biology techniques, as,for example, with polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant nucleotide sequences also includesynthetically derived nucleotide sequences, such as those generated, forexample, by using site-directed mutagenesis, but which still encode a Bttoxin receptor protein of the invention. Generally, variants of aparticular nucleotide sequence of the invention will have at least about40%, 50%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%,preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, andmore preferably at least about 98%, 99% or more sequence identity tothat particular nucleotide sequence as determined by sequence alignmentprograms described elsewhere herein using default parameters.

By “variant” protein is intended a protein derived from the nativeprotein by deletion (so-called truncation) or addition of one or moreamino acids to the N-terminal and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein; or substitution of one or more amino acidsat one or more sites in the native protein. Variant proteins encompassedby the present invention are biologically active, that is they continueto possess the desired biological activity of the native protein, thatis, activity as described herein (for example, Bt toxin bindingactivity). Such variants may result from, for example, geneticpolymorphism or from human manipulation. Biologically active variants ofa native Bt toxin receptor protein of the invention will have at leastabout 40%, 50%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%,preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, andmore preferably at least about 98%, 99% or more sequence identity to theamino acid sequence for the native protein as determined by sequencealignment programs described elsewhere herein using default parameters.A biologically active variant of a protein of the invention may differfrom that protein by as few as 1-15 amino acid residues, as few as 1-10,such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acidresidue.

The polypeptides of the invention may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants of the Bt toxin receptorpolypeptides can be prepared by mutations in the DNA. Methods formutagenesis and nucleotide sequence alterations are well known in theart. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S.Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques inMolecular Biology (MacMillan Publishing Company, New York) and thereferences cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al. (1978) Atlas ofProtein Sequence and Structure (Natl. Biomed. Res. Found., Washington,D.C.), herein incorporated by reference. Conservative substitutions,such as exchanging one amino acid with another having similarproperties, may be preferable.

Thus, the genes and nucleotide sequences of the invention include boththe naturally occurring sequences as well as mutant forms. Likewise, theproteins of the invention encompass both naturally occurring proteins aswell as variations and modified forms thereof. Such variants willcontinue to possess the desired toxin binding activity. Obviously, themutations that will be made in the DNA encoding the variant must notplace the sequence out of reading frame and preferably will not createcomplementary regions that could produce secondary mRNA structure. See,EP Patent Application Publication No. 75,444.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. For example, it is recognized that atleast about 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, and up to 960 amino acidsmay be deleted from the N-terminus of a polypeptide that has the aminoacid sequence set forth in SEQ ID NO:2, and still retain bindingfunction. It is further recognized that at least about 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 110, and up to 119 amino acids may be deletedfrom the C-terminus of a polypeptide that has the amino acid sequenceset forth in SEQ ID NO:2, and still retain binding function. Deletionvariants of the invention that encompass polypeptides having thesedeletions. It is recognized that deletion variants of the invention thatretain binding function encompass polypeptides having these N-terminalor C-terminal deletions, or having any deletion combination thereof atboth the C- and the N-termini.

However, when it is difficult to predict the exact effect of thesubstitution, deletion, or insertion in advance of doing so, one skilledin the art will appreciate that the effect will be evaluated by routinescreening assays. That is, the activity can be evaluated by receptorbinding and/or toxicity assays. See, for example, U.S. Pat. No.5,693,491; T. P. Keeton et al. (1998) Appl. Environ. Microbiol.64(6):2158-2165; B. R. Francis et al. (1997) Insect Biochem. Mol. Biol.27(6):541-550; T. P. Keeton et al. (1997) Appl. Environ. Microbiol63(9):3419-3425; R. K. Vadlamudi et al. (1995) J. Biol. Chem.270(10):5490-5494; Ihara et al. (1998) Comparative Biochem. Physiol. B120:197-204; Nagamatsu et al. (1998) Biosci. Biotechnol. Biochem.62(4):727-734, herein incorporated by reference.

Variant nucleotide sequences and polypeptides also encompass sequencesand polypeptides derived from a mutagenic and recombinogenic proceduresuch as DNA shuffling. With such a procedure, one or more differenttoxin receptor coding sequences can be manipulated to create a new toxinreceptor, including but not limited to a new Bt toxin receptor,possessing the desired properties. In this manner, libraries ofrecombinant polynucleotides are generated from a population of relatedsequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between the Bt toxinreceptor gene of the invention and other known Bt toxin receptor genesto obtain a new gene coding for a polypeptide with an improved propertyof interest, such as an increased ligand affinity in the case of areceptor. Strategies for such DNA shuffling are known in the art. See,for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech.15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al.(1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998)Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,448.

Where the receptor polypeptides of the invention are expressed in a celland associated with the cell membrane (for example, by a transmembranesegment), in order for the receptor of the invention to bind a desiredligand, for example a Cryl A toxin, the receptor's ligand binding domainmust be available to the ligand. In this aspect, it is recognized thatthe native Bt toxin receptor of the invention is oriented such that thetoxin binding site is available extracellularly.

Accordingly, in methods comprising use of intact cells, the inventionprovides cell surface Bt-toxin receptors. By a “cell surface Bt toxinreceptor” is intended a membrane-bound receptor polypeptide comprisingat least one extracellular Bt toxin binding site. A cell surfacereceptor of the invention comprises an appropriate combination of signalsequences and transmembrane segments for guiding and retaining thereceptor at the cell membrane such that that toxin binding site isavailable extracellularly. Where native Bt toxin receptors are used forexpression, deduction of the composition and configuration of the signalsequences and transmembrane segments is not necessary to ensure theappropriate topology of the polypeptide for displaying the toxin bindingsite extracellularly. As an alternative to native signal andtransmembrane sequences, heterologous signal and transmembrane sequencescould be utilized to produce a cell surface receptor polypeptide of theinvention.

It is recognized that it may be of interest to generate Bt toxinreceptors that are capable of interacting with the receptor's ligandsintracellularly in the cytoplasm, in the nucleus or other organelles, inother subcellular spaces; or in the extracellular space. Accordingly,the invention encompasses variants of the receptors of the invention,wherein one or more of the segments of the receptor polypeptide ismodified to target the polypeptide to a desired intra- or extracellularlocation.

Also encompassed by the invention are receptor fragments and variantsthat are useful, among other things, as binding antagonists that willcompete with a cell surface receptor of the invention. Such a fragmentor variant can, for example, bind a toxin but not be able to confertoxicity to a particular cell. In this aspect, the invention providessecreted receptors, more particularly secreted Bt toxin receptors; orreceptors that are not membrane bound. The secreted receptors of theinvention can contain a heterologous or homologous signal sequencefacilitating its secretion from the cell expressing the receptors; andfurther comprise a secretion variation in the region corresponding totransmembrane segments. By “secretion variation” is intended that aminoacids corresponding to a tranmembrne segment of a membrane boundreceptor comprise one or more deletions, substitutions, insertions, orany combination thereof; such that the region no longer retains therequisite hydrophobicity to serve as a transmembrane segment. Sequencealterations to create a secretion variation can be tested by confirmingsecretion of the polypeptide comprising the variation from the cellexpressing the polypeptide.

The polypeptides of the invention can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (i.e. recombinant) or synthesized using polypeptide synthesistechniques that are well known in the art. In one embodiment, thepolypeptide is produced by recombinant DNA methods. In such methods anucleic acid molecule encoding the polypeptide is cloned into anexpression vector as described more fully herein and expressed in anappropriate host cell according to known methods in the art. Thepolypeptide is then isolated from cells using polypeptide purificationtechniques well known to those of ordinary skill in the art.Alternatively, the polypeptide or fragment can be synthesized usingpeptide synthesis methods well known to those of ordinary skill in theart.

The invention also encompasses fusion polypeptides in which one or morepolypeptides of the invention are fused with at least one polypeptide ofinterest. In one embodiment, the invention encompasses fusionpolypeptides in which a heterologous polypeptide of interest has anamino acid sequence that is not substantially homologous to thepolypeptide of the invention. In this embodiment, the polypeptide of theinvention and the polypeptide of interest may or may not be operativelylinked. An example of operative linkage is fusion in-frame so that asingle polypeptide is produced upon translation. Such fusionpolypeptides can, for example, facilitate the purification of arecombinant polypeptide.

In another embodiment, the fused polypeptide of interest may contain aheterologous signal sequence at the N-terminus facilitating itssecretion from specific host cells. The expression and secretion of thepolypeptide can thereby be increased by use of the heterologous signalsequence.

The invention is also directed to polypeptides in which one or moredomains in the polypeptide described herein are operatively linked toheterologous domains having homologous functions. Thus, the toxinbinding domain can be replaced with a toxin binding domain for othertoxins. Thereby, the toxin specificity of the receptor is based on atoxin binding domain other than the domain encoded by Bt toxin receptorbut other characteristics of the polypeptide, for example, membranelocalization and topology is based on Bt toxin receptor.

Alternatively, the native Bt toxin binding domain may be retained whileadditional heterologous ligand binding domains, including but notlimited to heterologous toxin binding domains are comprised by thereceptor. Thus, the invention also encompasses fusion polypeptides inwhich a polypeptide of interest is a heterologous polypeptide comprisinga heterologous toxin binding domains. Examples of heterologouspolypeptides comprising Cryl toxin binding domains include, but are notlimited to Knight et al (1994) Mol Micro 11: 429-436; Lee et al. (1996)Appl Environ Micro 63: 2845-2849; Gill et al. (1995) J Biol Chem 270:27277-27282; Garczynski et al. (1991) Appl Environ Microbiol 10:2816-2820; Vadlamudi et al. (1995) J Biol Chem 270(10):5490-4, U.S. Pat.No. 5,693,491.

The Bt toxin receptor peptide of the invention may also be fused withother members of the cadherin superfamily. Such fusion polypeptidescould provide an important reflection of the binding properties of themembers of the superfamily. Such combinations could be further used toextend the range of applicability of these molecules in a wide range ofsystems or species that might not otherwise be amenable to native orrelatively homologous polypeptides. The fusion constructs could besubstituted into systems in which a native construct would not befunctional because of species specific constraints. Hybrid constructsmay further exhibit desirable or unusual characteristics otherwiseunavailable with the combinations of native polypeptides.

Polypeptide variants encompassed by the present invention include thosethat contain mutations that either enhance or decrease one or moredomain functions. For example, in the toxin binding domain, a mutationmay be introduced that increases or decreases the sensitivity of thedomain to a specific toxin.

As an alternative to the introduction of mutations, increase in functionmay be provided by increasing the copy number of ligand binding domains.Thus, the invention also encompasses receptor polypeptides in which thetoxin binding domain is provided in more than one copy.

The invention further encompasses cells containing receptor expressionvectors comprising the Bt toxin receptor sequences, and fragments andvariants thereof. The expression vector can contain one or moreexpression cassettes used to transform a cell of interest. Transcriptionof these genes can be placed under the control of a constitutive orinducible promoter (for example, tissue—or cell cycle-preferred).

Where more than one expression cassette utilized, the cassette that isadditional to the cassette comprising at least one receptor sequence ofthe invention, can comprise either a receptor sequence of the inventionor any other desired sequences.

The nucleotide sequences of the invention can be used to isolatehomologous sequences in insect species other than ostrinia, particularlyother lepidopteran species, more particularly other Pyraloidea species.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local homology algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the homology alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-similarity-method of Pearson and Lipman (1988) Proc. Natl.Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990)Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin and Altschul(1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0); the ALIGN PLUS program (version 3.0,copyright 1997); and GAP, BESTFIT, BLAST, FASTA, and TFASTA in theWisconsin Genetics Software Package, Version 8 (available from GeneticsComputer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignmentsusing these programs can be performed using the default parameters. TheCLUSTAL program is well described by Higgins et al. (1988) Gene73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al.(1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. TheALIGN and the ALIGN PLUS programs are based on the algorithm of Myersand Miller (1988) supra. A PAM120 weight residue table, a gap lengthpenalty of 12, and a gap penalty of 4 can be used with the ALIGN programwhen comparing amino acid sequences. The BLAST programs of Altschul etal (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin andAltschul (1990) supra. BLAST nucleotide searches can be performed withthe BLASTN program, score=100, wordlength=12, to obtain nucleotidesequences homologous to a nucleotide sequence encoding a protein of theinvention. BLAST protein searches can be performed with the BLASTXprogram, score=50, wordlength=3, to obtain amino acid sequenceshomologous to a protein or polypeptide of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0)can be utilized as described in Altschul et al. (1997) Nucleic AcidsRes. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used toperform an iterated search that detects distant relationships betweenmolecules. See Altschul et al. (1997) supra. When utilizing BLAST,Gapped BLAST, PSI-BLAST, the default parameters of the respectiveprograms (e.g., BLASTN for nucleotide sequences, BLASTX for proteins)can be used. See http://www.ncbi.hlm.nih.gov. Alignment may also beperformed manually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity using GAP Weight of 50 and LengthWeight of 3; % similarity using Gap Weight of 12 and Length Weight of 4,or any equivalent program. By “equivalent program” is intended anysequence comparison program that, for any two sequences in question,generates an alignment having identical nucleotide or amino acid residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by the preferred program.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, to find the alignment of two complete sequences that maximizesthe number of matches and minimizes the number of gaps. GAP considersall possible alignments and gap positions and creates the alignment withthe largest number of matched bases and the fewest gaps. It allows forthe provision of a gap creation penalty and a gap extension penalty inunits of matched bases. GAP must make a profit of gap creation penaltynumber of matches for each gap it inserts. If a gap extension penaltygreater than zero is chosen, GAP must, in addition, make a profit foreach gap inserted of the length of the gap times the gap extensionpenalty. Default gap creation penalty values and gap extension penaltyvalues in Version 10 of the Wisconsin Genetics Software Package forprotein sequences are 8 and 2, respectively. For nucleotide sequencesthe default gap creation penalty is 50 while the default gap extensionpenalty is 3. The gap creation and gap extension penalties can beexpressed as an integer selected from the group of integers consistingof from 0 to 200. Thus, for example, the gap creation and gap extensionpenalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e)(i) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%sequence identity, preferably at least 80%, more preferably at least90%, and most preferably at least 95%, compared to a reference sequenceusing one of the alignment programs described using standard parameters.One of skill in the art will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning, and thelike. Substantial identity of amino acid sequences for these purposesnormally means sequence identity of at least 60%, more preferably atleast 70%, 80%, 90%, and most preferably at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. However, stringent conditions encompasstemperatures in the range of about 1° C. to about 20° C. lower than theT_(m), depending upon the desired degree of stringency as otherwisequalified herein. Nucleic acids that do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides they encode are substantially identical. This may occur,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is when thepolypeptide encoded by the first nucleic acid sequence isimmunologically cross reactive with the polypeptide encoded by thesecond nucleic acid sequence.

(e)(ii) The term “substantial identity” in the context of a peptideindicates that a peptide comprises a sequence with at least 70% sequenceidentity to a reference sequence, preferably 80%, more preferably 85%,most preferably at least 90% or 95% sequence identity to the referencesequence over a specified comparison window. Preferably, optimalalignment is conducted using the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443-453. An indication thattwo peptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution. Peptides that are “substantially similar” share sequencesas noted above except that residue positions that are not identical maydiffer by conservative amino acid changes.

The nucleotide sequences of the invention can be used to isolatecorresponding sequences from other organisms, particularly otherinsects, more particularly other lepidopteran species. In this manner,methods such as PCR, hybridization, and the like can be used to identifysuch sequences based on their sequence homology to the sequences setforth herein. Sequences isolated based on their sequence identity to theentire Bt toxin receptor sequences set forth herein or to fragmentsthereof are encompassed by the present invention. Such sequences includesequences that are orthologs of the disclosed sequences. By “orthologs”is intended genes derived from a common ancestral gene and which arefound in different species as a result of speciation. Genes found indifferent species are considered orthologs when their nucleotidesequences and/or their encoded protein sequences share substantialidentity as defined elsewhere herein. Functions of orthologs are oftenhighly conserved among species.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism. The hybridization probes may be genomic DNA fragments,cDNA fragments, RNA fragments, or other oligonucleotides, and may belabeled with a detectable group such as ³²P, or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the Bt toxin receptorsequences of the invention. Methods for preparation of probes forhybridization and for construction of cDNA and genomic libraries aregenerally known in the art and are disclosed in Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.).

For example, the entire Bt toxin receptor sequence disclosed herein, orone or more portions thereof, may be used as a probe capable ofspecifically hybridizing to corresponding Bt toxin receptor sequencesand messenger RNAs. To achieve specific hybridization under a variety ofconditions, such probes include sequences that are unique among Bt toxinreceptor sequences and are preferably at least about 10 nucleotides inlength, and most preferably at least about 20 nucleotides in length.Such probes may be used to amplify corresponding Bt toxin receptorsequences from a chosen plant organism by PCR. This technique may beused to isolate additional coding sequences from a desired organism oras a diagnostic assay to determine the presence of coding sequences inan organism. Hybridization techniques include hybridization screening ofplated DNA libraries (either plaques or colonies; see, for example,Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.,Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Duration of hybridizationis generally less than about 24 hours, usually about 4 to about 12hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, N.Y.); and Ausubel et al., eds. (1995) Current Protocols inMolecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience,New York). See Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

Thus, isolated sequences that encode for a Bt toxin receptor protein andwhich hybridize under stringent conditions to the Bt toxin receptorsequences disclosed herein, or to fragments thereof, are encompassed bythe present invention. Such sequences will be at least about 40% to 50%homologous, about 60%, 65%, or 70% homologous, and even at least about75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or morehomologous with the disclosed sequences. That is, the sequence identityof sequences may range, sharing at least about 40% to 50%, about 60%,65%, or 70%, and even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

The compositions and screening methods of the invention are useful foridentifying cells expressing the BT toxin receptors of the invention,and variants and homologues thereof. Such identification could utilizedetection methods at the protein level, such as ligand-receptor binding;or at the nucleotide level. Detection of the polypeptide could be insitu by means of in situ hybridization of tissue sections but may alsobe analyzed by bulk polypeptide purification and subsequent analysis byWestern blot or immunological assay of a bulk preparation.Alternatively, receptor gene expression can be detected at the nucleicacid level by techniques well known to those of ordinary skill in anyart using complimentary polynucleotides to assess the levels of genomicDNA, mRNA, and the like. As an example, PCR primers complimentary to thenucleic acid of interest can be used to identify the level ofexpression. Tissues and cells identified as expressing the receptorsequences of the invention are determined to be susceptible to toxinswhich bind the receptor polypeptides.

Where the source of the cells identified to express the receptorpolypeptides of the invention is an organism, for example an insectplant pest, the organism is determined to be susceptible to toxinscapable of binding the polypeptides. In a particular embodiment,identification is in a lepidopteran plant pesr expressing the Bt toxinreceptor of the invention.

The invention encompasses antibody preparations with specificity againstthe polypeptides of the invention. In further embodiments of theinvention, the antibodies are used to detect receptor expression in acell.

In one aspect, the invention is particularly drawn to compositions andmethods for modulating susceptibility of plant pests to Bt toxins.However, it is recognized that the methods and compositions could beused for modulating susceptibility of any cell or organism to thetoxins. By “modulating” is intended that the susceptibility of a cell ororganism to the cytotoxic effects of the toxin is increased ordecreased. By “suceptibility” is intended that the viability of a cellcontacted with the toxin is decreased. Thus the invention encompassesexpressing the cell surface receptor polypeptides of the invention toincrease susceptibility of a target cell or organ to Bt toxins. Suchincreases in toxin susceptibility are useful for medical and veterinarypurposes in which eradication or reduction of viability of a group ofcells is desired. Such increases in susceptibility are also useful foragricultural applications in which eradication or reduction ofpopulation of particular plant pests is desired.

Plant pests of interest include, but are not limited to insects,nematodes, and the like. Nematodes include parasitic nematodes such asroot-knot, cyst, lesion, and renniform nematodes, etc.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Isolation of EC Bt Toxin Receptor

Standard recombinant methods well known to those of ordinary skill inthe art were carried out. For library construction, total RNA wasisolated from the midgut of European corn borer (ECB), Ostrinianubilalis. Corn borer larvae (for example, a mix of stage 2, 3, and 4,equal weight) can be pulverized in liquid nitrogen, homogenized, andtotal RNA extracted by standard procedures. PolyA RNA can be isolatedfrom the total RNA with standard PolyA isolation procedures, such as thePolyATact system from Promega Corporation, Madison, Wis. cDNA synthesiscan then be performed and, for example, unidirectional cDNA librariescan be constructed according to known and commercial procedures, such asthe ZAP Express cDNA synthesis kit from Stratagene, La Jolla, Calif.cDNA can be amplified by PCR, sized and properly digested withrestriction fragments to be ligated into a vector. Subcloned cDNA can besequenced to identify sequences with the proper peptide to identitycorresponding to published sequences. These fragments can be used toprobe genomic or cDNA libraries corresponding to a specific host, suchas Ostrinia nubilalis, to obtain a full length coding sequence. Probescan also be made based on Applicants disclosed sequences. The codingsequence can then be ligated into a desired expression cassette and usedto transform a host cell according to standard transformationprocedures. Such an expression cassette can be part of a commerciallyavailable vector and expression system; for example, the pET system fromNovagen Inc. (Madison, Wis.). Additional vectors that can be used forexpression include pBKCMV, pBKRSV, pPbac and pMbac (Stratagene Inc.),pFASTBacl (Gibco BRL) and other common bacterial, baculovirus,mammalian, and yeast expression vectors.

All vectors were constructed using standard molecular biology techniquesas described for example in Sambrook et al., (1989) Molecular Cloning: ALaboratory Manual (2^(nd) ed., Cold Spring Harbor Laboratory: ColdSpring Harbor, N.Y.).

Expression is tested by ligand blotting and testing for Bt toxinbinding. Ligand blotting, binding, and toxicity are tested by knownmethods; for example, as described in Martinez-Ramirez (1994) Biochem.Biophys. Res. Comm. 201: 782-787; Vadlarnudi et al. (1995) J Biol Chem270(10):5490-4, Keeton et al. (1998) Appl Environ Microbiol64(6):2158-2165; Keeton et al. (1997) Appl Environ Microbiol63(9):3419-3425; Ihara et al. (1998) Comparative Biochemistry andPhysiology, Part B 120:197-204; Nagamatsu et al. (1998) Biosci.Biotechnol. Biochem. 62(4):718-726 and Nagamatsu et al. (1998) Biosci.Biotechnol. Biochem. 62(4):727-734.

Identifying the CrylA(b) binding polypeptide in ECB was done by ligandblotting brush border membrane vesicle polypeptides and probing thosepolypeptides for binding with CrylA(b) toxin. Two polypeptides,approximately 210 and 205 kDa, were found to bind to CrylA(b). Blottingand binding were done essentially as described in the precedingparagraph.

Degenerate primers for RT-PCR were designed based on known Cryl toxinbinding polypeptide sequences from Manducca sexta and Bombyx mori. Theprimers are shown below. cDNA was constructed from total midgut RNA(cDNA synthesis kit GibcoBrL). Degenerate primers were used to amplifyproducts of the expected size. The annealing temperature used was 53° C.in generation of the 280 bp fragment and 55° C. when generating the 1.6kb fragment.

A 280 bp fragment was obtained from ECB midgut RNA. Upon cloning andsequencing, the fragment was identified as having homology with the Bttoxin receptor 1 polypeptide (BTR1) described in Vadlamudi et al. (1995)J Biol Chem 270(10):5490-4.

A similar approach was used to generate a 1.6 kilobase pair clone. Thesequence of primers used to generate the 280 base pair fragment were:

Primer BTRD1S: 5′GTTAMYGTGAGAGAGGCAGAYCC3′, (SEQ ID NO:8) and PrimerBTRD5A: 5′GGATRTTAAGMGTCAGYACWCCG3′. (SEQ ID NO:9)The sequence of primers used to generate the 1.6 kb fragment were:

Primer BTRD6S: (SEQ ID NO:10) 5′TCCGAATTCTTCTTYAACCTCATCGAYAACTT3′, andPrimer BTRD7A: (SEQ ID NO:11) 5′CGCAAGCTTACTTGGTCGATGTTRCASGTCAT3′

The 1.6 kb fragment clone was ligated in an E. coli expression vector,pET-28a-c(+), and expressed using the pET system (Novagen Inc., Madison,Wis.). Purified polypeptide encoded by this 1.6 kb fragment demonstratedbinding to CrylA(b) in ligand blots. An ECB midgut cDNA library wasgenerated and screened using this 1.6 kb clone, generating 120 positiveplaques. Thirty of these plaques were chosen for secondary screening andfifteen of those plaques were purified and sent for DNA sequencing.

The obtained nucleotide sequence of the selected Bt toxin receptor clonefrom ECB is set forth in SEQ ID NO: 1. The total length of the clone is5498 base pairs. The coding sequences are residues 162-5312. The CrylAbinding site is encoded by residues 4038-4547. The predictedtransmembrane domain is encoded by residues 4872-4928. The correspondingdeduced amino acid sequence for this Bt toxin receptor clone from ECB isset forth in SEQ ID NO: 2.

The purified polypeptide generated from the 1.6 kb fragment set forth inSEQ ID NO:7 was used to inoculate rabbits for the production ofpolyclonal antibodies. On zoo western blots prepared from brush bordermembrane vesicles from various insect species, this set of antibodiesspecifically recognized ECB Bt toxin receptor polypeptides, incomparison to Bt toxin receptor homologues polypeptides from otherinsect species. Rabbit polyclonal antibodies were also raised from apurified polypeptide corresponding to amino acids 1293-1462 of SEQ IDNO:2.

Example 2 Isolation of CEW and FAW Bt Toxin Receptor Orthologues

cDNA encoding a full-length Bt toxin receptor from corn earworm (CEW,Heliothis Zea) was isolated. The nucleotide sequence for this cDNA isset forth in SEQ ID NO: 3. Nucleotides 171-5360 correspond to the openreading frame. Nucleotides 4917-4973 correspond to the transmembraneregion. Nucleotides 4083-4589 correspond to the CrylA binding site. Thededuced corresponding amino acid sequence for the CEW Bt toxin receptoris set forth in SEQ ID NO: 4.

cDNA encoding a full-length Bt toxin receptor from fall armyworm (FAW,Spodoptera frugiperda) was isolated. The nucleotide sequence for thiscDNA is set forth in SEQ ID NO: 5. Nucleotides 162-5363 correspond tothe open reading frame. Nucleotides 4110-4616 correspond to the CrylAbinding site. Nucleotides 4941-4997 correspond to the transmembraneregion. Nucleotides 162-227 correspond to a signal peptide. The deducedcorresponding amino acid sequence for the FAW Bt toxin receptor is setforth in SEQ ID NO: 6.

Example 3 Binding and Cell Death in Lepidopteran Insect Cells Expressingthe Bt Toxin Receptors of the Invention

An in vitro system is developed to demonstrate the functionality of a Bttoxin receptor of the invention. The results disclosed in this exampledemonstrate that the ECB Bt toxin receptor of the invention (SEQ IDNOs:1 and 2) is specifically involved in the binding and killing actionof CrylAb toxin.

Well known molecular biological methods are used in cloning andexpressing the ECB Bt toxin receptor in Sf9 cells. A baculovirusexpression system (Gibco BRL Catalogue No. 10359-016) is used accordingto the manufacturer's provided protocols and as described below. S.frugiperda (Sf9) cells obtained from ATCC (ATCC-CRL 1711) are grown at27° C. in Sf-900 II serum free medium (Gibco BRL, Catalogue No.10902-088). These cells, which are not susceptible to CrylAb toxin, aretransfected with an expression construct (pFastBacl bacmid, Gibco BRLcatalogue NO. 10360-014) comprising an operably linked Bt toxin receptorof the invention (SEQ ID NO: 1) downstream of a polyhedrin promoter.Transfected Sf9 cells express the ECB Bt toxin receptor and are lysed inthe presence of CrylAb toxin. Toxin specificities, binding parameters,such as Kd values, and half maximal doses for cellular death and/ortoxicity are also determined.

For generating expression constructs, the ECB Bt toxin receptor cDNA(SEQ ID NO:1) is subjected to appropriate restriction digestion, and theresulting cDNA comprising the full-length coding region is ligated intothe donor plasmid pFastBac1 multiple cloning site. Followingtransformation and subsequent transposition, recombinant bacmid DNAcomprising the ECB Bt toxin receptor (RBECB1) is isolated. As a control,recombinant bacmid DNA comprising the reporter gene β-glucuronidase(RBGUS) is similarly constructed and isolated.

For transfection, 2 μg each RBECB1 or RBGUS DNA is mixed with 6 μl ofCellFectin (GibcoBRL catalogue NO. 10362-010) in 100 μl of Sf900 medium,and incubated at room temperature for 30 minutes. The mixture is thendiluted with 0.8 ml Sf-900 medium. Sf9 cells (10⁶/ml per 35 mm well) arewashed once with Sf-900 medium, mixed with the DNA/CellFectin mixture,added to the well, and incubated at room temperature for 5 hours. Themedium is removed and 2 ml of Sf-900 medium containing penicillin andstreptomycin is added to the well. 3-5 days after transfection, Westernblotting is used to examine protein expression.

For Western blotting, 100 μl of cell lysis buffer (50 mM Tris, pH7.8,150 mM NaCl, 1% Nonidet P-40) is added to the well. The cells arescraped and subjected to 16,000×g centrifugation. Pellet and supernatantare separated and subjected to Western blotting. An antibody preparationagainst ECB Bt toxin receptor (Example 1) is used as first antibody.Alkaline phosphatase-labelled anti-rabbit IgG is used as secondaryantibody. Western blot results indicate that the full length ECB Bttoxin receptor of the invention (SEQ ID NOs:1 and 2) is expressed in thecell membrane of these cells.

For determining GUS activity, the medium of the cells transfected withRBGUS is removed. The cells and the medium are separately mixed with GUSsubstrate and assayed for the well known enzymatic activity. GUSactivity assays indicate that this reporter gene is actively expressedin the transfected cells.

For determining toxin susceptibility, Cry toxins including but notlimited to CrylA, CrylB, CrylC, CrylD, CrylE, CrylF, CrylI, Cry2, Cry3,and Cry9 toxins (Schnepf E. et al. (1998) Microbiology and MolecularBiology Reviews 62(3): 775-806) are prepared by methods known in theart. Crystals are dissolved in pH 10.0, 50 mM carbonate buffer andtreated with trypsin. Active fragments of Cry proteins are purified bychromatography. Three to five days after transfection, cells are washedwith phosphate buffered saline (PBS). Different concentrations of activefragments of Cry toxins are applied to the cells. At different timeintervals, the cells are examined under the microscope to readilydetermine susceptibility to the toxins. Alternatively, cell death,viability and/or toxicity is quantified by methods well known in theart. See, for example, In Situ Cell Death Detection Kits available fromRoche Biochemicals (Catalogue Nos. 2 156 792, 1 684 809, and 1 684 817),and LIVE/DEAD® Viability/Cytotoxicity Kit available from MolecularProbes (catalogue No. L-3224).

A dose-dependent response of RBECB1-transfected cells to CrylAb isreadily observed, with determined Kd values well within the range formany receptors. Control cells, e.g. those transfected with pFastBac1bacmid without an insert or those transfected with RBGus are notsignificantly affected by CrylAb. Interaction with other Cry toxins aresimilarly characterized.

This in vitro system is not only be used to verify the functionality ofputative Bt-toxin receptors, but also used as a tool to determine theactive site(s) and other functional domains of the toxin and thereceptor. Furthermore, the system is used as a cell-based highthroughput screen. For example, methods for distinguishing live versusdead cells by differential dyes are known in the art. This allows foraliquots of transfected cells to be treated with various toxin samplesand to serve as a means for screening the toxin samples for desiredspecificity or binding characteristics. Since the system is used toidentify the specificity of Cry protein receptors, it is a useful toolin insect resistance management.

Example 4 Expression of the ECB Bt Toxin Receptor in Toxin SusceptibleStages of the Insect's Life Cycle

Total RNA was isolated from the eggs, pupae, adults, and the 1st throughthe 5th instar developmental stages, using TRIzol Reagent (Gibco BRL)essentially as instructed by the manufacturer.(Gibco BRL). The RNA wasquantitated and 20 ug of each sample was loaded onto a formaldehydeagarose gel and electrophoresed at constant voltage. The RNA was thentransferred to a nylon membrane via neutral capillary transfer andcross-linked to the membrane using ultraviolet light. For hybridization,a 460 base pair ECB Bt toxin receptor DNA probe (bases 3682 to 4141 inSEQ ID NO:1) was constructed from a 460 base pair fragment preparedaccording to the manufacturer's protocol for Amersham Rediprime IIrandom prime labeling system. The denatured probe was added to themembrane that had been prehybridized for at least 3 hours at 65° C. andallowed to incubate with gentle agitation for at least 12 hours at 65°C. Following hybridization, the membranes were washed at 65° C. for 1hour with 1/4×0.5M NaCl, 0.1M NaPO4 (ph 7.0), 6 mM EDTA and 1% SDSsolution followed by two 1 hour washes in the above solution withoutSDS. The membrane was air dried briefly, wrapped in Saran Wrap andexposed to X-ray film.

An ECB Bt toxin receptor transcript of 5.5 kilobase was expressedstrongly in the larval instars with much reduced expression in the pupalstage. The expression levels appeared to be fairly consistent from firstto fifth instar, while decreasing markedly in the pupal stage. Therewere no detectable transcripts in either the egg or adult stages. Theseresults indicate that the ECB Bt toxin transcript is being produced inthe susceptible stages of the insects life cycle, while not beingproduced in stages resistant to the toxic effects of CrylAb.

Example 5 Tissue and Subcellular Expression of the ECB Bt Toxin Receptor

Fifth instar ECB were dissected to isolate the following tissues: fatbody (FB), malpighian tubules (MT), hind gut (HG), anterior midgut (AM)and posterior midgut (PM). Midguts from fifth instar larvae were alsoisolated for brush border membrane vesicle (BBMV) preparation using thewell known protocol by Wolfersberger et al. (1987) Comp. Biochem.Physiol. 86A:301-308. Tissues were homogenized in Tris buffered saline,0.1% tween-20, centrifuged to pellet insoluble material, and transferredto a fresh tube. 50 ug of protein from each preparation was added to SDSsample buffer and B-mercaptoethanol, heated to 100° C. for 10 minutesand loaded onto a 4-12% Bis-Tris gel (Novex). After electrophoresis, theproteins were transferred to a nitrocellulose membrane using a semi-dryapparatus. The membrane was blocked in 5% nonfat dry milk buffer for 1hour at room temperature with gentle agitation. The primary antibody(Example 1) was added to a final dilution of 1:5000 and allowed tohybridize for 1 hour. The blot was then washed three times for 20minutes each in nonfat milk buffer. The blot was then hybridized withthe secondary antibody (goat anti-rabbit with alkaline phosphataseconjugate) at a dilution of 1:10000 for 1 hour at room temperature.Washes were performed as before. The bands were visualized by using thestandard chemiluminescent protocol (Tropix western light proteindetection kit).

The ECB Bt toxin receptor protein was only visible in the BBMV enrichedlane, and not detected in any of the other ECB tissues types. Thisresult indicates that the expression of the ECB Bt toxin receptorprotein is at very low levels, since the BBMV preparation is a 20-30fold enriched fraction of the midgut brush border. The result supportspropositions that the ECB Bt toxin receptor is an integral membraneprotein uniquely associated with the brush border. It also demonstratesthat the ECB Bt toxin receptor is expressed in the envisioned targettissue for CrylAb toxins. However, the result does not necessarily ruleout expression in other tissue types, albeit the expression of thisprotein in those tissues may be lower than in the BBMV enrichedfraction.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. An isolated Ostrinia nubialis insect receptor polypeptide havingBacillus thuringiensis (Bt) Cryl A toxin binding activity and an aminoacid sequence selected from the group consisting of: a) the amino acidsequence set forth in SEQ ID NO:2; b) an amino acid sequence having atleast 95% sequence identity to the amino acid sequence set forth in SEQNO:2; c) the amino acid sequence encoded by the nucleotide sequence setforth in SEQ ID NO:1; and, d) an amino acid sequence encoded by anucleotide sequence having at least 95% sequence identity to thenucleotide sequence set forth in SEQ ID NO:1.
 2. A fusion polypeptidecomprising the polypeptide of claim 1, and at least one additional Bttoxin receptor polypeptide.
 3. The fusion polypeptide of claim 2,wherein said additional Bt toxin receptor polypeptide has CrylA toxinbinding activity.